Targeted cancer therapies that can selectively kill cancer cells without harming other cells in the body would represent a major improvement in the clinical treatment of cancer. It would be highly desirable to develop a strategy to directly target cancer cells with chemotherapeutic agents in cancer treatment regimens. This could lead to reduction or elimination of toxic side effects, more efficient delivery of the drug to the targeted site, and reduction in dosage of the administered drug and a resulting decrease in toxicity to healthy cells and in the cost of the chemotherapeutic regimen. Reports of targeting chemotherapeutic drugs using antibodies have appeared in the literature since 1958. Targeting drugs by conjugation to antibodies for selective delivery to cancer cells has had limited success due to the large size of antibodies (MW=125-150 kilodaltons or KD) and thus their relative inability to penetrate solid tumors. An alternative strategy comprises the use of smaller targeting ligands and peptides, which recognize specific receptors unique to or overexpressed on tumor cells, as the targeting vector. Such constructs have molecular weights of 2-6 KD, which allow ready penetration throughout solid tumors.
Increased cell proliferation and growth is a trademark of cancer. The increase in cellular proliferation is associated with high turnover of cell cholesterol. Cells requiring cholesterol for membrane synthesis and growth may acquire cholesterol by receptor mediated endocytosis of plasma low density lipoproteins (LDL), the major transporter of cholesterol in the blood, or by de novo synthesis. LDL is taken up into cells by a receptor known as the LDL receptor (LDLR); the LDL along with the receptor is endocytosed and transported into the cells in endosomes. The endosomes become acidified and this releases the LDL receptor from the LDL; the LDL receptor recycles to the surface where it can participate in additional uptake of LDL particles. There is a body of evidence that suggests that tumors in a variety of tissues have a high requirement for LDL to the extent that plasma LDLs are depleted. The increased import of LDL into cancerous cells is thought to be due to elevated LDL receptors (LDLR) in these tumors. Some tumors known to express high numbers of LDLRs include some forms of leukemia, lung tumors, colorectal tumors and ovarian cancer. In vivo studies showed that LDLRs do appear in brain malignancies. Leppala et al used PET imaging, and demonstrated that 99mTc_LDL localizes in human brain tumors in vivo but not in normal brain.
This suggests that the LDL receptor is a potential unique molecular target in GBM and other malignancies for the delivery of anti-tumor drugs via LDL particles. A test of this possibility was undertaken by Maranhão and coworkers. A protein-free microemulsion (LDE) with a lipid composition resembling that of low-density lipoprotein (LDL) was used in metabolic studies in rats to compare LDE with the native lipoprotein. Incubation studies also showed that LDE incorporates a variety of apolipoproteins, including apo E, a ligand for recognition of lipoproteins by specific receptors.
Lipophilic Derivatives of Cancer Chemotherapeutic Agents:
Arbor Therapeutics has developed unique lipophilic derivatives of the cancer chemotherapeutic agent which have high stability in normal systemic circulation and retention in the lipid core of the LDL particles but readily release the active chemotherapeutic agent in the acidic environment of the endosome. See U.S. Pat. No. 8,440,714, the disclosure of which is incorporated herein in its entirety.
In another embodiment, there is provided a active chemotherapeutic compounds of the formula 3a or 3b:
wherein: R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is selected from O or S; Q is O or S; and T is O or S. In one aspect of the compound, R1 is hydrogen or C1-C4 alkyl; R2 is C5-C22 alkyl; Y is O or S; Z is O; Q is O; and T is O. The activated compound of the formula 3a or 3b may be used to prepare the acid labile lipophilic conjugate when the activated compound is condensed with a hydroxyl bearing cancer chemotherapeutic agent (HBCCA). As defined herein, the HBCCA is represented generically with the residue or group “R” in the formulae 1, 1a, 1b, 1.1, 2 and 2a, for example, and where the HBCCA is not coupled to form the acid labile, lipophilic molecular conjugates, then the HBCCA may also be generically represented as having the formula “R—OH” since the HBCCA may be functionalized by one or more hydroxyl (—OH) groups.
In one embodiment, there is provided an acid labile lipophilic molecular conjugate (ALLMC) of the formula 1, 1.1 or formula 2:
wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; for formula 1 or 1.1 R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is O or S; Q is O or S; and T is O or S; for formula 2: R2 is a C1-C22 alkyl; T is O or S; and X is hydrogen or a leaving group selected from the group consisting of mesylates, sulfonates and halogen (Cl, Br and I); and their isolated enantiomers, diastereoisomers or mixtures thereof, or a pharmaceutically acceptable salt thereof. The compound 1.1 includes the pure syn isomer, the pure anti isomer and mixtures of syn- and anti-isomers, and their diastereomers.
In another embodiment, there is provided the above acid labile lipophilic molecular conjugate of the formula 1 or 1.1 wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is O or S; Z is O; Q is O; and T is O. In one aspect of the acid labile lipophilic molecular conjugate of the formula 2 wherein: R2 is C5-C22 alkyl; T is O; and X is hydrogen or selected from the group consisting of Cl, Br and I. In another variation, R2 is C9-C22. In another aspect of the above acid labile lipophilic molecular conjugate comprising the formula 1a, 1b or formula 2a:
wherein: R is a hydroxyl bearing cancer chemotherapeutic agent (HBCCA); for formula 1a or 1b R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; and R2 is C5-C22 alkyl; and for formula 2a: R2 is C1-C22 alkyl; and X is hydrogen or is selected from the group consisting of Cl, Br and I. In one variation of the compound that is the carbonate (i.e., —OC(O)O—) of the formula 1a or 1b the compound is the corresponding sulfonate (i.e., —OS(O)O—) of the formula 1a wherein the carbonate group is replaced by a sulfonate group. The compound 1b includes the pure syn isomer, the pure anti isomer and mixtures of syn and anti isomers, and their diastereomers.
In another variation of the compound of the formula 1, 2, 1a and 2a, R1 is hydrogen or C1-C4 alkyl or C5-C22 alkyl, and R2 is the carbon residue of an unsaturated fatty acid, such as the carbon residue selected from the group consisting of the C19 residue of eicosenoic acid (including the cis isomer, trans isomer and mixtures of isomers), C17 residue of oleic acid and the C17 residue of elaidic acid. As used herein, the “carbon residue” (e.g., C17 residue, C19 residue etc. . . . ) of the fatty acid means the carbon chain of the fatty acids excluding the carboxyl carbon.
In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of taxanes, abeo-taxanes, camptothecins, epothilones, cucurbitacins, quassinoids, anthracyclines, and their analogs and derivatives. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, valrubicin and vinblastine.
In one embodiment, there is provided an acid labile lipophilic molecular conjugate (ALLMC) of the formula 1, 1.1 or formula 2:
wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; for formula 1 or 1.1 R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is O or S; Q is O or S; and T is O or S; for formula 2: R2 is a C1-C22 alkyl; T is O or S; and X is hydrogen or a leaving group selected from the group consisting of mesylates, sulfonates and halogen (Cl, Br and I); and their isolated enantiomers, diastereoisomers or mixtures thereof, or a pharmaceutically acceptable salt thereof. The compound 1.1 includes the pure syn isomer, the pure anti isomer and mixtures of syn- and anti-isomers, and their diastereomers.
In another embodiment, there is provided the above acid labile lipophilic molecular conjugate of the formula 1 or 1.1 wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is O or S; Z is O; Q is O; and T is O. In one aspect of the acid labile lipophilic molecular conjugate of the formula 2 wherein: R2 is C5-C22 alkyl; T is O; and X is hydrogen or selected from the group consisting of Cl, Br and I. In another variation, R2 is C9-C22. In another aspect of the above acid labile lipophilic molecular conjugate comprising the formula 1a, 1b or formula 2a:
wherein: R is a hydroxyl bearing cancer chemotherapeutic agent (HBCCA);for formula 1a or 1b R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; and R2 is C5-C22 alkyl; and for formula 2a: R2 is C1-C22 alkyl; and X is hydrogen or is selected from the group consisting of Cl, Br and I. In one variation of the compound that is the carbonate (i.e., —OC(O)O—) of the formula 1a or 1b the compound is the corresponding sulfonate (i.e., —OS(O)O—) of the formula 1a wherein the carbonate group is replaced by a sulfonate group. The compound 1b includes the pure syn isomer, the pure anti isomer and mixtures of syn and anti isomers, and their diastereomers.
In another variation of the compound of the formula 1, 2, 1a and 2a, R1 is hydrogen or C1-C4 alkyl or C5-C22 alkyl, and R2 is the carbon residue of an unsaturated fatty acid, such as the carbon residue selected from the group consisting of the C19 residue of eicosenoic acid (including the cis isomer, trans isomer and mixtures of isomers), C17 residue of oleic acid and the C17 residue of elaidic acid. As used herein, the “carbon residue” (e.g., C17 residue, C19 residue etc. . . . ) of the fatty acid means the carbon chain of the fatty acids excluding the carboxyl carbon. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of taxanes, abeo-taxanes, camptothecins, epothilones, cucurbitacins, quassinoids, anthracyclines, and their analogs and derivatives. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, valrubicin and vinblastine. In another aspect of the above acid labile lipophilic molecular conjugate, the conjugate is selected from the compounds in FIGS. 18, 19 and 20. In one variation, only one of the groups -ALL1, -ALL2, -ALL3 . . . to -ALLn is an -ALL group and the others are hydrogens. In another variation, two of the groups -ALL1, -ALL2, -ALL3 . . . to -ALLn are -ALL groups.
In another embodiment, there is provided a pharmaceutical composition comprising: a) a therapeutically effective amount of a compound of the above, in the form of a single diastereoisomer; and b) a pharmaceutically acceptable excipient. In another aspect, the pharmaceutical composition is adapted for oral administration; or as a liquid formulation adapted for parenteral administration. In another aspect, the composition is adapted for administration by a route selected from the group consisting of orally, parenterally, intraperitoneally, intravenously, intraarteriall, transdermally, intramuscularly, rectally, intranasally, liposomally, subcutaneously and intrathecally. In another embodiment, there is provided a method for the treatment of cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound or composition of any of the above compound or composition, to a patient in need of such treatment. In one aspect of the method, the cancer is selected from the group consisting of leukemia, neuroblastoma, glioblastoma, cervical, colorectal, pancreatic, renal and melanoma. In another aspect of the method, the cancer is selected from the group consisting of lung, breast, prostate, ovarian and head and neck. In another aspect of the method, the method provides at least a 10%, 20%, 30%, 40%, or at least a 50% diminished degree of resistance expressed by the cancer cells when compared with the non-conjugated hydroxyl bearing cancer chemotherapeutic agent.
In another embodiment, there is provided a method for reducing or substantially eliminating the side effects of chemotherapy associated with the administration of a cancer chemotherapeutic agent to a patient, the method comprising administering to the patient a therapeutically effective amount of an acid labile lipophilic molecular conjugate of the formula 1, 1.1 or formula 2:

wherein: R is a hydroxyl bearing cancer chemotherapeutic agent; for formula 1 or 1.1: R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is O or S; Q is O or S; and T is O or S; for formula 2: R2 is C1-C22 alkyl; T is O or S; and X is hydrogen or a leaving group selected from the group consisting of mesylates, sulfonates and halogen (Cl, Br and I); and their isolated enantiomers, diastereoisomers or mixtures thereof. The compound 1.1 includes the pure syn isomer, the pure anti isomer and mixtures of syn and anti isomers, and their diastereomers. In one variation of the above, R2 is C9-C22 alkyl. In one aspect, the method provides a higher concentration of the cancer chemotherapeutic agent in a cancer cell of the patient. In another aspect, the method delivers a higher concentration of the cancer chemotherapeutic agent in the cancer cell, when compared to the administration of a non-conjugated cancer chemotherapeutic agent to the patient, by at least 5%, 10%, 20%, 30%, 40% or at least 50%.
In another embodiment, there is provided a compound of the formula 3a or 3b:

wherein: R1 is hydrogen, C1-C4 alkyl or C5-C22 alkyl; R2 is C5-C22 alkyl; Y is selected from O, NR′ or S wherein R′ is hydrogen or C1-C6 alkyl; Z is selected from O or S; Q is O or S; and T is O or S. In one aspect of the compound, R1 is hydrogen or C1-C4 alkyl; R2 is C5-C22 alkyl; Y is O or S; Z is O; Q is O; and T is O. The activated compound of the formula 3a or 3b may be used to prepare the acid labile lipophilic conjugate when the activated compound is condensed with a hydroxyl bearing cancer chemotherapeutic agent (HBCCA). As defined herein, the HBCCA is represented generically with the residue or group “R” in the formulae 1, 1a, 1b, 1.1, 2 and 2a, for example, and where the HBCCA is not coupled to form the acid labile, lipophilic molecular conjugates, then the HBCCA may also be represented as having the formula “R—OH” since the HBCCA may be functionalized by one or more hydroxyl (—OH) groups.
Similarly, the acid labile lipophilic group (i.e., the “-ALL” group of the activated compound) that may be condensed with a HBCCA to form the acid labile, lipophilic molecular conjugate generically represented as “R-O-ALL.” Accordingly, where more than one -ALL group is condensed or conjugated with a HBCCA group, then each -ALL group may be independently designated as -ALL1, -ALL2, -ALL3 . . . to -ALLn where n is the number of available hydroxyl groups on the cancer chemotherapeutic agent that may be conjugated or couple with an -ALL group. As exemplified for the compound of formulae 1 and 2, for example, the HBCCA and the -ALL groups as designated, are shown below.

An example of an acid labile, lipophilic molecular conjugate (ALLMC), where the HBCCA group is paclitaxel having two -ALL groups, is depicted below:

In the above representative example of the acid labile molecular conjugate of paclitaxel, each of the -ALL1 and -ALL2 is independently hydrogen or an -ALL group as defined herein. For HBCCA groups having more than one hydroxyl groups, the inaccessible hydroxyl group or groups where the acid labile lipophilic group cannot be formed, then the group that is designated as an -ALL group(s) is hydrogen.
In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of taxanes, abeo-taxanes, camptothecins, epothilones, cucurbitacins, quassinoids, anthracyclines, and their analogs and derivatives. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, valrubicin and vinblastine.
Representative chemotherapeutic agents that may be employed in the present composition or formulations are disclosed in Figures A, B and C. In one aspect of the above, the chemotherapeutic agent is ART-207.

Capturing the great potential of selective and specific delivery of chemotherapeutic compounds to cancer tissues via their over expression of LDL receptors and consequent high uptake of LDL particles from the systemic circulation, requires that the cancer chemotherapeutic agent have high lipophilicity so as to remain entrapped in the lipid core of the LDL particle and not diffuse into the plasma to lead to toxic side effects from exposure of normal tissues to the agent. Further, once the LDL particle with its chemotherapeutic payload has entered the cancer cell via LDL receptor mediated uptake into the acidic environment of the endosome, the LDL receptor is disassociated from the LDL particle and is recycled to the cell surface and the LDL particle releases its lipid contents and its lipophilic chemotherapeutic agent to the enzymes and acidic environment of the endosome.
Further validity of this expectation was shown by Maranhão and coworkers who demonstrated that a cholesterol-rich microemulsion or nanoparticle preparation (LDE) concentrates in cancer tissues after injection into the bloodstream. The cytotoxicity, pharmacokinetics, toxicity to animals and therapeutic action of a paclitaxel lipophilic derivative associated to LDE were compared with those of commercial paclitaxel. Results showed that LDE-paclitaxel oleate was stable. The cytostatic activity of the drug in the complex was diminished compared with the commercial paclitaxel due to the cytotoxicity of the vehicle Cremophor EL used in the commercial formulation. Competition experiments in neoplastic cultured cells showed that paclitaxel oleate and LDE are internalized together by the LDL receptor pathway. Tolerability to mice was remarkable, such that the lethal dose (LD50) was nine fold greater than that of the commercial formulation (LD50=326 μM and 37 μM, respectively). LDE concentrates paclitaxel oleate in the tumor roughly fourfold relative to the normal adjacent tissues. At equimolar doses, the association of paclitaxel oleate with LDE resulted in remarkable changes in the drug pharmacokinetic parameters when compared to commercial paclitaxel (t1/2=218 mm and 184 mM, AUC=1,334 μg-h/mL and 707 μg-h/mL and CL=0.125 mL/min and 0.236 mL/min, respectively). The therapeutic efficacy of the complex was pronouncedly greater than that of the commercial paclitaxel, as indicated by the reduction in tumor growth, increase in survival rates and % cure of treated mice. Maranhão et al showed LDE-paclitaxel oleate is a stable complex and compared with paclitaxel, toxicity is considerably reduced and activity is enhanced which may lead to improved therapeutic index in clinical use. Maranhão and coworkers followed up their preliminary animal studies with a pilot clinical study in breast cancer patients. The clinical study was performed in breast cancer patients to evaluate the tumoral uptake, pharmacokinetics and toxicity of paclitaxel associated to LDE nanoemulsions. Twenty-four hours before mastectomy 3H-paclitaxel oleate associated with 14C-cholesteryl oleate-nanoemulsion or 3H-paclitaxel in Cremophor EL were injected into five patients for collection of blood samples and fragments of tumor and normal breast tissue. A pilot clinical study of paclitaxel-nanoemulsion administered at 3-week intervals was performed in four breast cancer patients with refractory advanced disease at 175 and 220 mg/m2 dose levels. The half-life (t1/2) of paclitaxel oleate associated to the nanoemulsion was longer than that of paclitaxel (t1/2=15.4±4.7 and 3.5±0.80 h, respectively). Uptake of the 14C-cholesteryl ester nanoemulsion and 3H-paclitaxel oleate by breast malignant tissue was threefold greater than the normal breast tissue and toxicity was minimal at the two dose levels. Their results suggest that the paclitaxel-nanoemulsion preparation can be advantageous for use in the treatment of breast cancer because the pharmacokinetic parameters are improved, the drug is concentrated in the neoplastic tissue and the toxicity of paclitaxel is reduced. Additional reports from the Maranhão laboratory of small human trials with the LDL-like lipid emulsion show that lipophilic drugs incorporated into the core of the emulsion are targeted to tumor tissue and side effects are significantly reduced. The difficulty of preparation of the emulsion, manufacture by long term sonication and extended centrifugation for particle size selection precluded them from further clinical exploration and development.
We have discovered how to prepare a nanoparticulate “pseudo LDL” lipid microemulsion as a delivery formulation for sufficiently lipophilic chemotherapeutics, including our unique acid labile, lipophilic prodrug derivative of the cancer chemotherapeutic agent. In one embodiment, the lipophilic chemotherapeutic agents have a measured or calculated LogP of greater than 4. We further demonstrate in animal tumor models that the acid labile, lipophilic molecular conjugates of cancer chemotherapeutic agent when dosed in a nanoparticulate, LDL-like lipid emulsion, is more useful for tumor reduction due to reduced toxicity and greater efficacy due to selective delivery to neoplastic/tumor tissue.
In one embodiment, the application discloses a stable, synthetic low density lipoprotein (LDL) nanoparticle comprising: a) a lipophilic anti-cancer agent; b) phospholipids (PL); and c) triglycerides (TG); wherein the LDL nanoparticle has a particle size less than 100 nm, less than 90 nm or less than 80 nm. As referred to herein, a stable synthetic low density lipoprotein (LDL) nanoparticle is a nanoparticle as defined herein that has a shelf life at about 25° C. of greater than 90 days, greater than 120 days, greater than 180 days, or greater than 1 year when stored in a sealed container and away from exposure to light. In another aspect, the nanoparticle has a shelf life at about 25° C. that is more than 1 year, or about 2 years or more when stored in a sealed container and away from exposure to light. In one aspect if the LDL nanoparticle, the particle size distribution is between 40 to 80 nm. In another aspect, the particle size distribution is between 50 and 60 nm. In one aspect of the above, the LDL nanoparticle has a mean size distribution of 60 nm. In another aspect, the LDL nanoparticle has a mean size distribution of about 50 nm. In another aspect, the phospholipids is selected from the group consisting of phosphotidylcholine, phosphotidylethanolamine, symmetric or asymmetric 1,2-diacyl-sn-glycero-3-phosphorylcholines, 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine,1,2-dimyristoyl-sn-glycero-3-phosphorylethanolamine, egg phospholipids, egg phosphatidyl glycerol, dipalmitoylphosphatidylglycerol, egg lecithin, soy lecithin, lecithin (NOS) and mixtures thereof.
In another aspect, the LDL nanoparticle further comprises cholesterol ester (CE) or cholesterol (C), or mixtures of cholesterol ester and cholesterol. In another aspect, the cholesterol ester is selected from the group consisting of C16-22 esters of cholesterol, cholesterol and mixtures thereof; and the triglycerides is selected from the group consisting of soybean oil, triolein, glyceryl tripalmitate and mixtures thereof. In one aspect of the above, the esters of cholesterol is selected from the group consisting of cholesteryl oleate, cholesteryl palmatate, cholesteryl stearate and cholesteryl lenolenate. In another aspect, the LDL nanoparticle further comprises an agent selected from the group consisting of triolein, natural antioxidants, BHT, ubiquinol, ubiquinol 10, vitamin E, alpha-tocopherol, gamma-tocopherol, lycopene, retinyl derivative and betacarotene, or mixtures thereof. In another aspect, the lipophilic anti-cancer agent is an anti-cancer agent or a prodrug of the anti-cancer agent. In one aspect of the above, the ratio of PL:TG may range from 8:1 to 3:1. In another aspect, the ratio of PL:TG:CE may range from 8:1:0.5 to 3:1:0.1. In another aspect, the ratio of the lipophilic anti-cancer agent: PL:TG may range from 1:10:3 to 1:3:0.5. In another aspect, the ratio of the lipophilic anti-cancer agent: PL:TG:CE may range from 1:10:3:1 to 1:3:0.5:0.1.
In another aspect, the anti-cancer agent is selected from the group consisting of a taxane, abeo-taxane, camptothecin, epothilone, cucurbitacin, quassinoid and an anthracycline. In another aspect, the anticancer agent is selected from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, valrubicin and vinblastine. In yet another aspect, the pro-drug of the anti-cancer agent is an acid labile lipophilic molecular conjugates is as disclosed herein, and in Figures A, B and C. In one particular aspect, the acid labile lipophilic molecular conjugates is ART-207.

In another aspect of the above, the lipophilic anti-cancer agent has a logP greater than 4.0, 6.0 or 8.0. In one aspect, the weight ratio of PL:TG:CE:C ranges from 73:12:2:1 to 78:12:2:1; optionally further comprising an additive selected from the group consisting of triolein, natural antioxidants, BHT, ubiquinol, ubiquinol 10, vitamin E, alpha-tocopherol, gamma-tocopherol, lycopene, retinyl derivative and betacarotene, or mixtures thereof. In one variation, the weight ratio of PL:TG:CE:C is 77:10:2:1. In one aspect, the natural antioxidant is selected from Coenzyme Q10, resveratrol, pterostilbene and mixtures thereof. In another aspect, the ratio of the lipophilic anti-cancer agent to the triglyceride is from 1:1 to 0.6:1. In another aspect, the LDL nanoparticle contains a total solids content of 6.0 to 8% wt/wt. In another aspect, the LDL nanoparticle contains a total lipid content of 5.0 to 7.0% wt/wt. In one variation, the LDL nanoparticle further comprises a poloxamer selected from the group consisting of P188, P237, P338, P407, SYNPERONICS, PLURONICS and KOLLIPHOR, or mixtures thereof.
In another embodiment, there is provided a process for preparing a stable, synthetic low density lipoprotein (LDL) nanoparticle comprising: a) a lipophilic anti-cancer agent; b) phospholipids (PL); and c) triglycerides (TG); the process comprising: 1) combining the lipophilic anti-cancer agent, phospholipids and triglycerides to form a mixture; 2) homogenizing the mixture by dissolution in a volatile solvent; 3) removing the solvent; 4) forming a coarse emulsion by blending of the mixture in a buffer to form an emulsion mixture; 5) microfluidizing the emulsion mixture in a microfluidizer apparatus for a sufficient amount of time to produce a particle preparation of 100 nm or less; and 6) sterilizing the nanoparticle preparation through a 0.22 micron filter to obtain the synthetic LDL nanoparticles with a range of 40 nm to 80 nm. In one variation, the synthetic low density lipoprotein (LDL) nanoparticle mixture wherein the phospholipids is selected from the group consisting of phosphotidylcholine, phosphotidylethanolamine, symmetric or asymmetric 1,2-diacyl-sn-glycero-3-phosphorylcholines, 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine,1,2-dimyristoyl-sn-glycero-3-phosphorylethanolamine, egg phospholipids, egg phosphatidyl glycerol, dipalmitoylphosphatidylglycerol, egg lecithin, soy lecithin, lecithin (NOS) and mixtures thereof. In another aspect, the LDL nanoparticle further comprises cholesterol (C) or cholesterol ester (CE) selected from the group consisting of C16-22 esters of cholesterol, cholesterol and mixtures thereof; and the triglycerides is selected from the group consisting of soybean oil, triolein, glyceryl tripalmitate and mixtures thereof; or mixtures of cholesterol and cholesterol esters. In one aspect of the above, the slow speed blending is performed at a speed of between 200 and 800 rpm, about 200 rpm, 400 rpm, 600 rpm or 800 rpm. In another aspect, the microfluidizing of the warm coarse emulsion mixture is performed at a processing temperature of about 45 to 65° C. In another aspect of the above process, the solvent is removed in vacuum. In another embodiment, there is provided a stable, synthetic low density lipoprotein (LDL) nanoparticle comprising: a) a lipophilic anti-cancer agent; b) phospholipids (PL); and c) triglycerides (TG) prepared by the process as disclosed herein. In one embodiment of the above, the synthetic LDL nanoparticle is prepared by any of the disclosed process, wherein the LDL nanoparticle becomes coated with apolipoprotein upon intra venous injection and are recognized and internalized by cellular LDL receptors.
In another embodiment, there is provided a method for the treatment of cancer in a patient comprising administering to the patient a therapeutically effective amount of the stable, synthetic low density lipoprotein (LDL) nanoparticle of any one of the above embodiments, aspects and variations, to a patient in need of such treatment. In another aspect of the method, the cancer is selected from the group consisting of leukemia, neuroblastoma, glioblastoma, cervical, colorectal, pancreatic, renal melanoma, lung, breast, prostate, ovarian and head and neck.
Development of lipid-based Drug and Pro-Drug formulations:
Optimization of Drug/ProDrug incorporation capacity, particle size and stability. General procedures for preparation of nanoparticulate lipid based “pseudo LDL” formulations are found in Arbor Therapeutics, LLC Standard Operating Procedures; ART 001 Coarse Emulsion Preparation Rev. 1, ART 002 Microfluidics Model 110P Gen II (MF) Rev 1, and ART 003 Nicomp 380 ZLS Particle Size Analyses Rev. 1. Exceptions to these SOPs are noted.
Abbreviations: PC—phosphatidylcholine, TG—triglycerides, TC—total cholesterol, FC—free cholesterol, CE—esterified cholesterol, U—Ubiquinol, VitE—Vitamin E (mixed tocopherols), P188—Poloxamer 188, DMPC—1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, PS—Phosphotidyl serine; MFP—microfluidizer processing, IC—interaction chamber, ICJ—interaction chamber jacket; TSPM—total solids premix; REM—resultant emulsion; %—percent of total solids, TL, %—percent of total lipids, W/V—weight to volume; Recovery, %—percent of ART-207 recovered in formulation after microfluidizer processing and sterile 0.22 um filtration; mfg—manufacturing; Pre-mix—pre-mixture; ND—not determined; BDL—below detection limit. R—Resistance.
All of the logP calculations for the anti-cancer agents noted in the table below were done online at www.chemicalize.org which uses the logP predictor from ChemAxon. The ChemAxon algorithm is based on: Vellarkad N. Viswanadhan et al., Journal of Chemical Information and Computer Sciences 1989 29 (3), 163-172.
Anti-CancerAnti-CancerAgentslogPApprovedAgentsLogPApprovedART-15324.61n/aART-1376.22ART-16412.91n/aMitotane6.11AdrenalART-15212.86n/aEpothilone D5.10ART-20912.56n/aEpothilone C4.85ART-16312.19n/aMasoprocol4.76ART-15112.04n/aCabozantinib4.66ThyroidART-20711.68n/aLapatinib4.64BreastART-15611.42n/aART-4484.53ART-18511.40n/aValrubicin4.49BladderART-16111.31n/aART-2874.41ART-46711.15n/aImatinib4.38LeukemiasART-20811.05n/aSorafenib4.34Liver, Kidney,ThyroidART-16210.69n/aEpothilone4.21ART-4499.77n/aCabazitaxel4.20ProstateART-4419.32n/aCarfilzomib4.20MultiplemyelomaGossypol8.02Vinblastine4.18Breast,myeloma,testicularFulvestrant7.57BreastAxitinib4.15KidneyEverolimus7.40KidneyEpothilone B4.12ART-3327.19Bosutinib4.09CMLTemsirolimus7.13KidneyMaterials used:
ReagentVendorPart NumberLot NumberPhosphatidylcholineLipoid, GMBHLipoid E 801032718-121052Mfg Date July 2010Retest Date July 2013DMPS (1,2-Dimyristoyl-sn-Glycero-Avanti Polar Lipids, Inc.840033P140PS-833-[Phospho-L-Serine) (Sodium Salt)Soybean Oil (triglycerides)CriscoPure Vegetable Oil1095420Soybean oilBest Before 5 APR. 2013Cholesterol, 95%Alfa AesarA11470L23W024Cholesterol Oleate (cholesterol ester)Alfa AesarA-11378G03Y033Vitamin E, mixed tocopherolsSwanson (AMD)SW152Illegible Lot #, Mfg DateJuly 2012Ubiquinol, co-enzyme QKaneka CorporationKaneka QH ™FB02-0104Polaxamer P-188 (Pluronic F68;Spectrum ChemicalsP 11692AK0895Polyethylene-Polypropylene Glycol)CAS 90003-11-6N.F.DMPC (1,2-dimyristoyl-sn-glycero-Avanti Polar Lipids, Inc.850345P140PC-2613-phosphorylcholine)ART-207Arbor Therapeutics, LLCART-207AW001- 243AW004-13AW004-24ART 287Arbor Therapeutics, LLCART 287Sodium Chloride, ACSAlfa Aesar12314L12X071Distilled WaterKroger GroceryDistilled WaterNAMethylene Chloride, ACS StabilizedFisher ScientificL-14119125544NitrogenNexAirHP NI-33904713-10-4Glacial Acetic Acid, ACSReagents, Inc.5-10060-31004NESterile Filters, 0.22 μm, PESFisher Scientific50 mL 09-741-88NA150 mL 09-741-01NA500 mL 09-761-107NACuvettes, polystyrene 4.5 mLFisher Scientific14 955 125NAEquipment used:
DescriptionManufacturerModel Number400 gram balanceDenver InstrumentSI-403100 gram balanceDenver InstrumentAPX-100Emersion Hand BlenderOster ®2605Variable Voltage ControlGlas-Col ®104A PL1202MicroFludizer ®MicroFluidics, Inc.M110P GEN IIParticle SizerParticle Sizing SystemsNicomp ™ 380 ZLSHPLCAgilentHP 1100 SeriesAnalytical Quantitation of ART-207:
The Analytical Method Development for Quantitation of ART-207 concentration in LDL like lipid emulsion nanoparticles was an evolutionary process over approximately 18 months.
Summary of Methods Used and Method Changes:
Taxane_Prodrug.M—Variable sized neat emulsion injections:
13 AUG. 2012Calibration Curve/Response Linearity17 JAN. 2013Dilution of emulsion samples 1:10 with IPATaxane_Test.M—Dilution of emulsion samples 1:10 with IPA, 1 μL injections:
Rev. 020 JAN. 2013Higher column temperature 55° C. vs 40° C.Rev. 19 FEB. 2013External Standard Preparation change to100 mg/100 mLRev. 211 MAR. 2013Injection Volume increase from 1 μL to 3 μLRev. 310 MAY 2013Use of bracketing external standards andduplicate sample analyses defined in draftStandard Operating Procedure -ART005, HPLC Analysis of Formulated ART-207Methods used to Quantitate ART-207 in emulsions prepared for the following studies:
Taxane_Prodrug.M9 OCT. 2012In-Vitro StudyTaxane Test.M, Rev. 020 & 21 JAN. 2013MTD StudyTaxane Test.M, Rev. 16 MAR. 2013MTD StudyTaxane Test.M, Rev. 213 MAR. 2013Efficacy StudyTaxane Test.M, Rev. 313 MAY 2013PK/PD and Particle SizeComparison Toxicity/Efficacy Study
The three HPLC Methods used were similar. Emulsion samples were injected neat during this time. All methods used a Phenomenex 4.6×50 mm Luna 5μ C18(2) 100A, part number 00B-4252-E0 column, flow rate: 1.5 mL/minute, detection: 230 nm, column temperature: 40° C., and injection volume: variable.
The gradient tables and injection volumes for each of these three methods are as follows:
B = % 0.01 M A = Acetonitrile H3PO4 WaterTaxane_Prodrug_7 Taxane_Prodrug.MTaxane_Prodrug_Short.Mmin.MInjection VolumeInjection VolumeInjection Volume2 μL1 μL20 μLMin.% A% BMin.% A% BMin.% A% B0505005050050501505015050150503100031000310001010006100071000115050750508505013505095050105050
Taxane_Prodrug.M was used to determine linearity of response which was shown to be R2=0.9997 for ART-207 from 0.6 to 5.2 mg/mL.
Taxane_Prodrug.M was also used to quantitate the emulsion prepared for the In-Vitro Cytotoxicity Study.
All Taxane_Prodrug.M methods showed carryover of emulsion components from a previous injection to the subsequent injections. Part of this carryover co-eluted with the ART-207 peak. Buffer blanks between samples helped a little. A 3 minute column wash method using 100 μL injections of 50/50 chloroform/methanol reduced the carryover considerably. Analyses of drug free formulations for the MTD Study showed no drug presence in the analyzed samples that was not possible to demonstrate with previous analytical procedures. Blank subtractions were not appropriate since emulsions had a peak eluting as the same time as ART-207.
A new method was developed which solved the emulsion component carryover problem. The higher column temperature 55° C. vs 40° C. may assist in liquefying and dissolving the emulsion particles better, allowing them to be washed off the column in the column wash. The gradient starts at higher acetonitrile concentration and the gradient is shallower to provide for resolution of any impurities. Analyses of drug free emulsions may be performed with confidence in a “Below Detection Limit” statement of result.
The HPLC analytical method used to quantitate ART-207 in lipid emulsions is TAXANE TEST.M, Rev. 0, performed on an Agilent 1100 quaternary pump and single wavelength system. The column is a Phenomenex 4.6×50 mm Luna 5μ C18(2) 100A, part number 00B-4252-E0. The method conditions are: flow rate: 1.5 mL/minute, detection: 230 nm, column temperature: 55° C., and injection volume: 1 μL. The gradient table is as follows:
Time, minutes% Acetonitrile% 0.01M H3PO4 Water075257100091000107525117525
The typical retention time of ART-207 is ±5.8 minutes in this method. Lipid emulsion sample preparation is 1 part emulsion into 9 parts isopropanol (1:10). Linearity of response was shown to be R2=0.9997 for ART-207 from 0.6 to 5.2 mg/mL in a similar acetonitrile/water C18 method. Quantitation is accomplished by using a response factor calculated from an external standard. The data are shown in the table. During the preparations of the lipid emulsions for this MTD study, unexplained fluctuations in the concentration of ART-207 were observed and investigated. The external standards were prepared using approximately 1 mg of ART-207 dissolved in 1 mL of solvent. Accuracy and consistency were improved when the external standard preparation was changed to 100 mg of ART-207 dissolved in 100 mL of solvent (Taxane Test.M, Rev. 1). Taxane Test.M, Rev. 1 method was used to re-determine and revise concentrations of ART-207 in emulsions prepared for the MTD study. The values reported initially (Taxane Test.M, Rev. 0) and the more accurate re-determined values (Taxane Test.M, Rev. 1) are shown in the table below.
Taxane Test.M, Rev. 0Taxane Test.M, Rev. 1Original ConcentrationRecalculated Concentration,Lot NumberReported, mg/mLmg/mL002-119-12.012.26002-119-23.023.40002-119-33.984.49002-119-46.136.91
On 11 Mar. 2013 the injection volume was increased from 1 μL to 3 μL to reduce the impact of injection to injection variability on the standard area counts used to determine response factor as well as sample area counts, Taxane Test.M, Rev. 2.
Bracketing external standards and duplicate analyses of samples were used in analytical quantitation of ART-207 and are defined in Standard Operating Procedure—ART 005, HPLC Analysis of Formulated ART-207, Taxane Test.M, Rev. 3.
Analytical Quantitation of 287 (Lot# ISI-30052013-1).
Taxane Test.M, Rev 3″ Analytical Method was used for Quantitation of ART 287 concentration in LDL like lipid emulsion nanoparticles. Sterile 0.22 um filtration of resultant emulsions did not significantly affect the ART-207 content in experiments described below. The particle size for all prepared coarse suspensions was always in the range 400-800 nm and did not affect the rate of particle size decrease during MF processing. MF processing volume was 100 ml unless specified. For all examples described below, see also Master Tables 1, 2 and 3.
Experiment 1. Preparation of Drug-Free Lipid Emulsion Using Original Formulation
To investigate effect of discrete passes vs recycling mode (return of the material into the feed reservoir) and controlled (≤60° C.) ICJ temperature on particle size.
TABLE 1aFormulation composition.Components Weighed, mg (per 100 ml)DateLot#MaterialPCTGFCCEUVit EARTP188DMPCPS17 Dec. 2012002.102.1TSPM2330123920615310100000
TABLE 1bRatios of major formulation components.TG/ART-207PC/ART-207PC/TGFC/CEN/AN/A1.881.35Coarse suspension was prepared and MF processed (lot#002.103.1).
TABLE 1cParticle size, ART-207 content, and particle stability of resultant emulsion.Particle sizeART-207Formulation StabilityManufacturingby intensity,Content,Days pastParticle sizeDateLot#nmmg/mlRecovery, %mfgnm18 Dec. 2012002.103.163N/AN/A3679
MFP. FIG. 1 shows that particle size reaches the 55-60 nm plateau after 40 discrete passes that is equal to 20 min of processing (one discrete pass is ˜30 sec). MFP was stopped after 80 passes and material filtered. Particle size slightly increased after 0.22 μm filtration from 55 to 63 nm Processing via discrete passes at ˜60-65° C. (temperature of IC jacket) did not result in improvement of particle size relative to lipid formulations generated in prior studies. Particle size analysis and stability. The resultant emulsion was unstable. In FIG. 2 and Table 1c, particle size increased from 63 to 79 nm over 36 days.
Experiment 2. Preparation of drug-free lipid emulsion using original formulation.
Investigate the effect of lower (<30° C.) ICJ temperature (i.e. effect of “local” cooling of IC jacket) on resultant particle size.
TABLE 2aFormulation composition.Components Weighed, mg (per 100 ml)DateLot#MaterialPCTGFCCEUVit EARTP188DMPCPS17 Dec. 2012002.102.2TSPM2338124720315210100000
TABLE 2bRatios for major formulation components.TG/ART-207PC/ART-207PC/TGFC/CEN/AN/A1.871.34
Coarse emulsion was prepared and MF processed (lot#002.104.1). MF processing was performed in recycling mode.
TABLE 2cParticle size, ART-207 content, and particle stability of resultant emulsion.Particle sizeART-207Formulation StabilityManufacturingby intensity,Content,Days pastParticle sizeDateLot#nmmg/mlRecovery, %mfgnm19 Dec. 2012002.104.145N/AN/A3582
2a. Ice cubes were placed around IC jacket. After 10 min of MF processing the particle size dropped from 439 nm (coarse emulsion) to 66 nm, and in 20 min it reached a plateau at ˜58 nm. Further processing to 30 mm did not decrease the particle size (FIG. 3).
2b. To achieve better contact of cooling agent with IC and to further lower its temperature, ice was removed from lower tray and glycol bath set to 10° C. was used to submerge interaction chamber (with surrounding pipelines and back pressure chamber) in 10° C. glycol. These cooling conditions allowed further decreasing of the particle size to 43 nm (FIG. 3).
Particle size analysis of drug-free formulation. In FIG. 4, Table 1c, and 2c, the resultant particle size was significantly smaller after processing at <30° C. temperatures compare to processing at 60° C. Both processing conditions resulted in unstable particles. 63 to 79 and 45 to 82 nm particle size increase was observed in lot#002.103.1 and 002.104.1, respectively.
Experiment 3. Preparation of ART-207 containing lipid emulsion using original formulation. Investigating ART-207 (Lot# AW-001-243) incorporation capacity of original formulation and effect of ART-207 on particle size and stability.
TABLE 3aFormulation composition.Components Weighed, mg (per 100 ml)DateLot#MaterialPCTGFCCEUVitEARTP188DMPCPS18 Dec. 2012002.103.2TSPM217611451841411010320000
TABLE 3bRatio for major formulation components.TG/ART-207PC/ART-207PC/TGFC/CE3.586.801.901.30
Coarse suspension was prepared and MF processed (lot#002.105.1).
TABLE 3cParticle size, ART-207 content, and particle stability of resultant emulsion.Particle sizeART-207Formulation StabilityManufacturingby intensity,Content,Days pastParticle sizeDateLot#nmmg/mlRecovery, %mfgnm19 Dec. 2012002.105.1823.1498.135196
MFP. In FIG. 5, particle size reaches the plateau or resistance (Resistance 1-R1) at ˜130 nm after 30 mm of processing at 10-20° C. Raising the temperature to 50-60° C. resulted in lowering particle size to 100 nm and reaching R2. Lowering the temperature back to 10-20° C. resulted in further particle size decrease to 77 nm. MFP was stopped and material was filtered. Slight increase of particle size from 77 to 82 nm was observed after filtration.
HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane_Prodrug.M) was 3.14 mg/ml. Data indicate that 98% of the drug used for preparation of this formulation was incorporated into lipid particles (Table 3c).
Particle size analysis of ART-207 containing formulation. The resultant ART-207 containing emulsion was unstable. FIG. 6 and Table 3c show that particle size increased from 82 to 196 nm over 35 days.
Addition of ART-207 to formulation notably decreases stability of resultant emulsion.
Experiment 4. Preparation of ART-207 (Lot# AW-001-243) containing lipid emulsion. Investigating the effect of increased phospholipid content and decreased FC/CE ratio while keeping the amount of TC the same (table 4b and 4a) on drug incorporation capacity and stability of resultant nanoparticles.
TABLE 4aFormulation composition.Components Weighed, mg (per 100 ml)DateLot#MaterialPCTGFCCEUVit EARTP188DMPCPSJan. 9, 2013002.107.2TSPM41571397703471010648000
TABLE 4bRatios for major formulation components.TG/ART-207PC/ART-207PC/TGFC/CE2.166.422.980.20Coarse suspension was prepared and MF processed (lot#002.108.1).
TABLE 4cParticle size, ART-207 content, and particle stability of resultant emulsion.Particle sizeART-207Formulation StabilityManufacturingby intensity,Content,Days pastParticle sizeDateLot#nmmg/mlRecovery, %mfgnm9 Jan. 2013002.108.11294.8174.214184
MFP. In FIG. 7, particle size reaches R1 at ˜125 nm after 40 mm of processing at ˜60° C. Lowering the temperature to ˜20° C. resulted in lowering particle size to 80 nm and reaching R2. Raising the temperature to 50° C. resulted in particle size increase to ˜108 nm. MFP was stopped and filtered. Increase of particle size from 108 to 129 nm was observed after filtration.
HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane_Prodrug.M) was 4.81 mg/ml. Data indicate that 74% of the drug used for preparation of this formulation was incorporated into lipid particles (Table 4c). Particle size analysis of ART-207 containing formulation. The resultant ART-207 containing emulsion was unstable. In FIG. 8 and Table 4c, particle size increased from 129 to 184 nm over 14 days.
Increased phospholipid content and decreased FC/CE ratio resulted in higher ART-207 particle content but did not improve stability of resultant emulsion.
Experiment 5. Preparation of ART-207 (Lot# AW-001-243) containing lipid emulsion.
Investigate effect of further increase of phospholipid concentration and decrease of CE on drug incorporation capacity and stability of resultant nanoparticles; and B. To optimize the temperature control strategy: raising the temperature from 20° C. to 60° C. in experiment #4 resulted in undesirable increase of the particle size.
TABLE 5aFormulation composition.Components Weighed, mg (per 100 ml)DateLot#MaterialPCTGFCCEUVit EARTP188DMPCPS9 Jan. 2013002.108.2TSPM50251397712161010648000
TABLE 5bRatios for major formulation components.TG/ART-207PC/ART-207PC/TGFC/CE2.167.753.600.33Coarse suspension was prepared and MF processed (lot#002.109.1).
TABLE 5cParticle size, ART-207 content, and particle stability of resultant emulsion.Particle sizeART-207Formulation StabilityManufacturingby intensity,Content,Days pastParticle sizeDateLot#nmmg/mlRecovery, %mfgnm9 Jan. 2013002.109.1745.5084.933169
MFP. In FIG. 9, particle size reaches R1 at ˜105 nm after 30 min of processing at ˜60° C. Lowering the temperature to ˜20° C. resulted in lowering particle size to 73 nm and reaching R2. MFP was stopped and filtered. No increase of particle size was observed after filtration.
HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane_Prodrug.M) was 5.5 mg/ml. Data indicate that 85% of the drug used for preparation of this formulation was incorporated into lipid particles (Table 5c). The resultant drug content of the particles was higher than in previous experiment indicating that increase of coating material (phospholipid) facilitates drug incorporation (see Tables 3a, 3c, 4a, and 4c).
Particle size analysis of ART-207 containing formulation. Although the resultant particle size was significantly smaller (Table 5c) compared to previous experiment (Table 4c), the emulsion was extremely unstable. In FIG. 10 and Table 5c, particle size increased from 74 to 149 nm over 14 days and from 74 to 169 nm over 33 days.
Further increase of phospholipid and decrease of CE and subsequently TC content resulted in smaller particles and higher (relative to previous experiment #4) capacity of the formulation to incorporate ART-207 but did not improve stability of resultant emulsion. A repeat of this experiment gave similar results which indicate the processing parameters are reproducible and give reproducible outcomes.
Experiment 6. Preparation of ART-207 (Lot# AW-001-243) containing lipid emulsion. Increased ART-207 content results in decreased stability of the resultant particles, experiment #6 was performed to determine the effect of lowering ART-207 concentration on particle size and stability.
TABLE 6aFormulation composition.Components Weighed, mg (per 100 ml)DateLot#MaterialPCTGFCCEUVit EARTP188DMPCPS10 Jan. 2013002.109.3TSPM51701392702131010213000
TABLE 6bRatio for major formulation components.TG/ART-207PC/ART-207PC/TGFC/CE6.5424.273.710.33
Coarse suspension was prepared and MF processed (lot#002.111.1).
TABLE 6cParticle size, ART-207 content, and particle stability of resultant emulsion.Particle size ART-207Formulation StabilityManufacturingby intensity, Content,Recovery,Days pastParticle size, DateLot#nmmg/ml%mfgnm11 Jan. 2013002.111.1471.8687.32466MFP. In FIG. 11, particle size reaches R1 at ˜66 nm after 20 mm of processing at ˜60° C. Lowering the temperature to ˜20° C. resulted in lowering particle size to 42 nm MFP was stopped and was filtered. Increase of particle size from 42 to 47 nm was observed after filtration.
HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane_Prodrug.M) was 1.86 mg/ml. Data indicate that 87% of the drug used for preparation of this formulation was incorporated into lipid particles (Table 6c). Particle size analysis of ART-207 containing formulation. The resultant ART-207 containing emulsion was more stable relative to previous formulations. In FIG. 12 and Table 6c, particle size increased from 47 to 66 nm over 24 days.
Lowering ART-207 and maintaining high phospholipid content (Table 6a and b) resulted in: a) significantly smaller particle size compared to the size previously attained in experiment #5 (see also tables 5a,b,c and 6a,b,c), and b) more stable particles (relative to emulsions obtained in previous experiments).
Experiment 7. Preparation of ART-207 (Lot# AW-001-243) containing lipid emulsion.
Effect of lowering triglycerides content on ART-207 incorporation, particle size and stability.
TABLE 7aFormulation composition.Components Weighed, mg (per 100 ml)DateLot#MaterialPCTGFCCEUVit EARTP188DMPCPS11 Jan. 2013002.110.4TSPM5900652691501010648000
TABLE 7bRatios for major formulation components.TG/ART-207PC/ART-207PC/TGFC/CE1.019.109.050.46
Coarse suspension was prepared and MF processed (lot#002.111.2).
TABLE 7cParticle size, ART-207 content, and particle stability of resultant emulsion.Particle size ART-207Formulation StabilityManufacturingby intensity,Content,Recovery, Days pastParticle sizeDateLot#nmmg/ml%mfgnm11 Jan. 2013002.111.2663.8859.93875
MFP. In FIG. 13, particle size reached R1 at ˜105 nm after 20 mm of processing at ˜60° C. Lowering the temperature to ˜20° C. and further to 10° C. resulted in further increase of the particle size from 105 to 118 nm and reaching R2. Increasing temperature back to 50-60° C. resulted in particle size decrease to 90 nm and reaching next resistance point at 94 nm. Additional 30 mm processing at ˜˜25° C. decreased particle size from 94 to 84 nm MFP was stopped and filtered. Decrease of particle size from 84 to 66 nm was observed after filtration.
HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane_Prodrug.M) was 3.88 mg/ml. Data indicate that 60% of the drug used for preparation of this formulation was incorporated into lipid particles (7c). ART-207 incorporation capacity of the particles with low triglycerides content was decreased. Particle size analysis of ART-207 containing formulation. The ART-207 containing emulsion was more stable compared to formulations higher in triglycerides processed in experiments #3-6. In FIG. 14 and Table 7c, particle size did not increase from 66 to 75 nm over 38 days. For the next 30 days (68 days total) particle size increased to 92 nm.
Thus, lowering triglyceride content and maintaining high phospholipid content resulted in reasonably small and fairly stable particles. ART-207 incorporating capacity of low triglycerides emulsion was significantly decreased. Increase of phoshpolipid results in smaller particles with increased ART-207 content; Increase of phospholipid does not improve the particle stability; Decrease of ART-207 content results in decrease of particle size and improved stability; Decrease of TG content and therefore, TG/ART-207 ratio from ˜2 to 1 results in decreased particle size, improved stability, but lower ART-207 incorporating capacity of formulation.
TG/ART-207 ratio appears to be an important pre-requisite in determining ART-207 incorporation capacity of the formulation; Higher TG/ART-207 ratio results in increased ART-207 incorporation capacity of the formulation but compromised particle stability, and lower ratio results in improved formulation stability but lower ART-207 incorporation capacity. There are at least two possible routes to optimizing ART-207 incorporation, particle size, and stability of emulsion preparations: Optimizing ratios for major formulation components.
Experiment 8. Preparation of ART-207 (Lot# AW-001-243) containing emulsion.
Investigating the effect of Poloxamer P188 (1% V/W) addition on ART-207 incorporation, particle size and stability. Poloxamers are non-ionic poly (ethylene oxide) (PEO)-poly (propylene oxide) (PPO) copolymers. Poloxamers are broadly used in clinical applications (1). Ability of P188 to intercalate in lipid monolayers and to seal the membranes (2) suggests their usefulness in improving stability of the lipid particles by possibly relieving the surface tension. P188 coating of nanoparticles reduces their opsonization by serum proteins and macrophage uptake (3) that is particularly relevant for in vivo applications. One of the most relevant features of P188 to this application is that P188 coating of the lipid nanoparticles does not prevent binding of Apolipoprotein E (4).